The formation of hydroxyl radical spin adduct of DMPO was observed during the reaction of SOD and SOD mutants (G37R and H46R) with hydrogen peroxide. The signal intensity of the hydroxyl radical spin adduct of DMPO obtained with WT-SOD was comparable to G37R/hydrogen peroxide but no spin adduct was observed with H46R/hydrogen peroxide. SOD and FALS SOD mutants were constructed by replacement of Cys6 by Ala and Cys111 by Ser. A similar trend was obtained by following the oxidation of ABTS to ABTS cation radical. In the present study we also show that ABTS cation radical itself can react with DMPO or PBN and this reaction could lead to erroneous interpretation of results. This reaction occurred at a much faster rate with DMPO than with PBN. The quantification of copper in three SOD proteins was studied by X-band ESR at 77 K. The amount of copper bound varied as follows: wild>G37R>>H46R. Incubation of SOD with hydrogen peroxide in chelex-treated PBS resulted in an ESR spectrum that is composed of two copper species with different coordination sites. The X-band ESR spectrum of WT-SOD obtained at liquid nitrogen temperature is characteristic of an axially symmetric copper coordination site (a-parallel = 145 G; g-parallel = 2.26 and g-perpendicular = 2.06). We conclude that the coordination of copper changes from four histidine residues to a different environemtn possibly consisting of histidine and two oxygen residues.